力学和电刺激联合作用对大鼠骨髓间充质干细胞向心肌细胞方向分化调控的研究

Bone marrow-derived mesenchymal stem cells (BMSCs) are a potential source of material for the generation of tissue-engineered cardiac grafts because of their ability to transdifferentiate into cardiomyocytes after chemical treatments or co-culture with cardiomyocytes. More recent evidences suggest that the physical microenvironment can also govern stem cell differentiation. For example, the previous results of our group have shown that both cyclic strain and fluid shear stress could affect the cardiomyogenic differentiation of BMSCs. However, in those in vitro experiments, only a very small proportion of MSCs showed a cardiomyocyte phenotype, so their potential clinical benefits for cardiac repair are quite limited. The electrical signal is essential for the development and function of heart. The electrophysiological properties of cardiomyocytes are determined by the uneven distribution and transmembrane movement of the intracellular and extracellular ions. Many researchers have reported that electrical stimulation could influence the behaviors and functions of osteoblasts, endothelial cells, nerve cells, cardiomyocytes and stem cells. Cardiomyocytes in the body are constantly experiencing physical stimuli, including cyclic mechanical stretch, shear atress and electrical stimulation. In a search for new methods to induce MSC to differentiate into cardiomyocytes for cardiac therapies and to gain a better understanding of the role of physical stimuli in cardiovascular development and remodeling, the project intends to investigate the effects of combined mechanical loading and electrical stimulation on cardiomyogenic differentiation of BMSCs and the possible mechanism of ion channels using the cellular strain-electrical and shear stress-electrical loading devices developed and patented by our laboratory. In addition, this work has far-reaching implications in terms of MSC differentiation under physical stimuli in bioreactors, and provides a basis for the design of new in vitro cell culture systems for stem cell and tissue engineering.

骨髓间充质干细胞是用于心肌组织工程和细胞治疗的重要细胞，在生物化学因素的诱导下可以向心肌细胞分化，新近许多研究表明物理因素也可以影响干细胞的分化，如本项目前期结果发现周期性牵张和剪切能够诱导干细胞向心肌细胞方向分化，但仍存在诱导分化方法有限及分化效率低等问题。心脏发育及功能产生过程中电信号是必不可少的，细胞内外各种离子的不均匀分布及其跨膜运动决定了心肌细胞的电生理特性，电刺激能影响成骨细胞、内皮细胞、神经细胞、心肌细胞和干细胞的生物学行为和功能。结合国内外相关的最新报道，考虑到体内心肌细胞同时处于力和电的影响下，拟利用本实验室自主研制的反应器对大鼠骨髓间充质干细胞加载牵张-电刺激和剪切-电刺激，研究力-电联合作用对干细胞向心肌细胞方向分化的影响，探讨离子通道在这一过程中的可能作用机制，有助于丰富干细胞分化调控理论，为组织工程种子细胞的研究应用和生物反应器的合理设计提供实验依据和理论基础。

心血管疾病对人类生命的威胁日益严峻，来源于干细胞的心肌细胞移植疗法有望成为心肌损伤性疾病的最有潜力的治疗方法，其中骨髓间充质干细胞因具有多向分化潜能及避免伦理争议等优点，成为了研究热点之一。干细胞所处微环境影响的研究已从化学和生物方面开始聚焦于物理因素对干细胞分化的作用，如申请人和其他实验室的前期研究表明对间充质干细胞施加牵张或剪切应力可以影响其向心肌细胞方向的分化效率，而电刺激也可以促进这一过程。进一步提高间充质干细胞的诱导分化效率，促进其定向分化，实现对干细胞的分化控制是决定干细胞未来应用的关键。. 本项目首先研究了单独电刺激对大鼠骨髓间充质干细胞分化的影响。对大鼠骨髓间充质干细胞加载了不同形式和大小的电刺激，结果表明单独的电刺激组并没有出现心肌相关因子的表达，电刺激可以促进5氮胞苷或心肌细胞裂解液诱导的大鼠骨髓间充质干细胞向心肌细胞方向的分化，这可以从心肌细胞相关因子的基因和蛋白的表达方面得到证实。然后，本项目重点研究了力学因素和电刺激联合作用对大鼠骨髓间充质干细胞向心肌细胞方向分化的影响。利用本实验室自主研制并拥有专利的生物反应器对大鼠骨髓间充质干细胞加载牵张-电刺激和剪切-电刺激，研究结果表明力电联合刺激比力或电单独作用可以更好地诱导大鼠骨髓间充质干细胞表达更多的心肌细胞相关因子，尤其是牵张-电刺激联合作用组。另外，本项目还初步探讨了离子通道在这一过程中的可能作用。最后，超出申请书计划，本项目研究了磁场对大鼠骨髓间充质干细胞向心肌细胞方向分化的影响，以及初步探索了力学因素对诱导性多能干细胞向心肌细胞方向分化的影响。. 本项目已发表期刊论文2篇，另有论文3篇在整理撰写中；发表国际会议论文1篇，国内会议论文4篇；培养硕士研究生2名，另有2名硕士研究生即将毕业。本项目有助于丰富干细胞分化调控理论，为组织工程种子细胞的研究应用和生物反应器的合理设计提供参考。